2006 Fiscal Year Final Research Report Summary
Explanation of partner proteins forming complex with PHGPx of rat testis
| Project/Area Number |
17570158
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | University of Yamanashi |
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Principal Investigator |
YOKOTA Sadaki University of Yamanashi, Interdisciplinary Graduate School of Medicine and Engineering, Prof., 大学院医学工学総合研究部, 教授 (40020755)
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| Project Period (FY) |
2005 – 2006
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| Keywords | PHGPx / Rat testis / complex / mitochondria / Blue native PAGE |
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| Research Abstract |
Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenium-containing enzyme whose major function is reduction of esterified phospholipid or peroxidized cholesterol of membrane. The enzyme is expressed in various tissues among which testis shows the highest expression. Previously we have clarified that PHGPx localizes to various subcellular compartments of rat spermatogenic cells. On the other hand, it has been proved that our peptide antibody against 15 amino acid chain at the C-terminus of human PHGPx dose not react with PHGPx in testis extract without heating and reduction of the extract. This fact suggests that PHGPx forms protein complex so that the C-terminal region is masked by binding protein. We suppose that PHGPx might have not only anti-oxidant function but also other unknown functions considering the variety of subcellular localizations and the complex formation of PHGPx. Then, we isolated active protein complexes from rat testicular mitochondrial fraction by blue native polyacrylamide electrophoresis (BN-PAGE) and analyzed the isolated complexes by SDS-PAGE and Western blotting. We found at first time that rat testicular mitochondrial PHGPx was associated with other proteins to form complexes and could be dissociated to 19 kDa PHGPx by heating and reduction, showing that binding of PHGPx with other proteins was non-covalent. The size of the complexes varied over 400 kDa to 100 kDa. This wide range of the complex size might reflect heterogeneity of the mitochondria derived from various spermatogenic cells with different developing stage. In addition, the variety of the complexes suggests different functions of the PHGPx complexes in the spermatogenic cells with each developing stage. We tried to determine partners of PHGPx by PMF analysis of subunit spots dissociated the complexes but we did not success. In next step of study, we purify each complex, analyze the subunit spots by PMF and specify the partner proteins.
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